PROJECT SUMMARY Cisplatin is one of the most effective anti-cancer drugs employed for the treatment of various solid tumors. Nevertheless, patients relapse because of the development of cisplatin resistance leading to therapeutic failure. Many studies have challenged the model of chemoresistance, yet the precise mechanism is still elusive. To better understand the role of protein kinase signaling in cisplatin resistance, we used a kinome-wide small hairpin RNA library to screen for a kinase, which has synthetic lethality when targeted with cisplatin in human cancers, including head/neck, lung and ovarian cancers. We found that suppression of microtubule associated serine/threonine kinase 1 (MAST1) selectively rescued the cisplatin sensitivity in cisplatin-resistant cancer cells, leading to decreased cell proliferation, apoptosis induction, and cell cycle arrest in a kinase-dependent manner. Moreover, MAST1 knockdown and cisplatin treatment achieved synergistic tumor growth inhibition in vivo in xenograft mice. Through a phosphorylation profiling, we identified MEK1 as a novel downstream substrate of MAST1. Mechanistic studies revealed that cisplatin inhibits the MAPK pathway by dissociating cRaf from MEK1, while MAST1 binds and phosphorylates MEK1 to reactivate the MAPK pathway. In addition, through a comprehensive proteomics study and transcription factor profiling, we identified upstream regulators of MAST1. In particular, we identified Hsp90AB1 as a binding partner and a stabilizer of MAST1, and glucocorticoid receptor (GR) as a potential transcription factor of MAST1. Furthermore, our preliminary clinical studies revealed that MAST1 upregulation positively correlates with cisplatin resistance in primary tumor tissues from head/neck cancer patients treated with cisplatin, suggesting MAST1 signaling pathway as a promising prognostic marker and a therapeutic target to treat human cancers in combination with cisplatin. Therefore, we screened and identified a FDA approved small molecule kinase inhibitor lestaurtinib as a potent MAST1 inhibitor and a cisplatin-sensitizing agent. Our central hypothesis is that MAST1 provides cisplatin-resistant proliferative signals through its potential substrate and effectors in cancer cells. Thus, MAST1 signaling represents a promising anti-cancer target in combination with cisplatin. We will use head and neck cancer as a research platform. Three specific aims are proposed: (1) To decipher the molecular mechanism underlying cisplatin-induced MAST1-MEK1 activation, which confers cisplatin resistance in human cancer; (2) To determine how MAST1 is induced and stabilized by GR and Hsp90AB1, respectively to mediate cisplatin resistance; (3) To validate MAST1 as a therapeutic target in treatment of cisplatin-resistant cancer using the newly identified MAST1 inhibitor lestaurtinib.